Name: in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)
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Description: Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

作者要感谢过去的库存实验室的成员:铭敏李凯瑟琳Plichta，维多利亚米兰达 - 汤普森和萨姆 - 金圭他们对许多这些协议的改进作出贡献。这项工作是由美国国家科学基金会资助的IOS-0840932和IOS-0724978 SP的股票投资部分

1 在体内的线虫的饲养与他们Symboitic细菌<ol><li>翻转100×15毫米的塑料培养皿中，并将滤纸（90 MM）两张碟的菜的盖子。 </li><li>均匀分布1毫升的IJ（感染性幼虫）的水悬浮液（在1000-2000 IJ / ml浓度）在滤纸上。 注:IJS不需要进行表面灭菌。 </li><li>添加更多的waxmoth 大蜡螟的10末龄幼虫的菜。的目标是提供大约100-200 IJS /幼虫。 </li><li>盖上培养皿（ 图1）的底部的盖子和标签的培养皿中。何况下面的信息:线虫种类名称（如已知）;隔离代码/名称;最新感染的陷阱设置。 </li><li>里面放置一个松散的密封塑料袋的菜（以保存水分），并保持它在黑暗中，在室温或在20-25℃的恒温箱。检查每天的菜 注:黑暗便于线虫感染，因为它模仿自然感染状况，在土壤中。 </li><li> 3-5天后，取出尸体线虫感染的迹象，以修改后的白色陷阱7。 注意:如果尸体腐烂的气味，这可能是一个迹象表明，培养不成功和/或有污染。有一批新的线虫和昆虫树立了新的传染箱。 </li></ol> 2， 体外线虫的饲养与他们共生菌<ol><li>肝肾阴虚型琼脂法9,19 <ol><li>斩肝肾小块（2 毫升或更小），并放入用NaCl，琼脂搅拌机及300ml水并混合，直到肉变成液体浆，厚浆或果浆。然后，转移混合物至1升的锥形烧瓶中。 </li><li>添加最终200毫升水，以搅拌机容器中，并弃去剩余的存储介质插入锥形烧瓶中。高压釜中进行15分钟，在121℃。可高压灭菌后的琼脂冷却浇注前触及。 注:中移液不建议，因为这种媒介往往有大块的肉。 </li><li>倒〜20毫升琼脂到5（或6）厘米的培养皿中，同时用手搅拌或纷飞的锥形瓶中，注入了更均匀的介质。让琼脂在4℃下固化和存储菜肴，直到需要 注意:使用火焰消毒金属铲，打破了任何大块的肉倒入培养皿中。 </li></ol></li><li>脂琼脂法9,19 <ol><li>通过营养肉汤，琼脂和酵母提取物混合在一起制备脂质琼脂培养基倒入混合到2升的锥形烧瓶中。加蒸馏水H 2 O和氯化镁2•6H 2 O和高压灭菌15分钟，在121℃。 </li><li>添加玉米油和玉米糖浆混合无菌混合，搅拌或摇动剧烈，经常均匀地分散油滴。 <br/&gt;注:通过将所需量的紫外线交联剂消毒玉米油和糖浆。中石油将不会溶解到液体中，但要确保它是在微小液滴。 </li><li>倒〜20毫升琼脂到5（或6）厘米的培养皿中，并允许琼脂在4°C，直到需要巩固和存储菜肴。 </li><li>数所需的共生细菌到脂质琼脂平板上，孵育板在28℃，24〜48小时。 注:价差100-200微升过夜（12-16小时）的LB亚文化。 </li><li>一天后，加入约表面消毒线虫悬浮液（或鸡蛋感染幼体）0.4毫升培养板在室温下在黑暗中或在孵化器在22±3℃。 注意:不要添加超过0.4毫升线虫悬浮液，因为它可以创建一个过于潮湿的环境，不利于线虫生长。 </li><li>日常监测的文化。文化应该生产出新一代IJS在12天左右（ 图2）。</li><li>转底菜用琼脂时，线虫看到爬行的菜（ 图3A）的两侧，以修改后的白色陷阱（见奥罗斯科等 12）收获IJS（ 图3B）。执行下在层流罩此步骤，以降低介质的污染。 </li><li>出苗后，收集修改的白色陷阱IJS和冲洗IJ停牌，以消除任何媒体碎片。商店IJ在无菌蒸馏水在10℃下（在冷温度下孵化或冷室中）或立即使用，如果需要的。 注:根据本研究的目的，与种子或者共生，殖民或aposymbiotic线虫板。 </li></ol></li></ol> 3， 体外 Aposymbiotic的饲养（共生免费）昆虫病原线虫<ol><li>感染10-20 G.蜡螟幼虫所需的线虫种类IJS（见第1节）。后3或4天（此时IJS应该已经成熟第一代成虫）收集尸体。 注:到期日，并需要获得卵子足够数量将种不同的女性数量。感染多G。蜡螟幼虫如果有必要，并早在48小时后感染，以评估他们是否在正确的发展阶段开始解剖。 </li><li>单独将每个尸体在培养皿用生理盐水溶液（M9缓冲18）。拉它的头用细尖镊子解剖尸体。 </li><li>收集至少20妊娠（里面的蛋）女性用细针（L型最好），并放置在含有10毫升M9的缓冲区（ 图4A）手表玻璃。 </li><li>通过考虑下列成分制备axenizing解决方案:6.75蒸馏水，1.25毫升5N的NaOH和2.25毫升市售漂白水稀释成3％的次氯酸盐。 注:氢氧化钠是碱性溶液，手套，护目镜和其他适当的保护衣服应穿。 注:准备新鲜axenizing溶液各一次，如漂白剂降解的光。准备axenizing液多批次，并在多个钟表眼镜进行表面灭菌的鸡蛋和收获。 </li><li>冲洗女性至少三次，M9缓冲填充表玻璃用缓冲溶液，吸干缓冲用移液管。确保女性留在表玻璃。重复此步骤两次。 </li><li>立即添加axenizing溶液并让女性保持在该溶液中不超过10-15分钟以上。观察线虫组织开始崩解，而鸡蛋（它们是耐axenizing溶液）保持不变。 </li><li>手动扰乱女性的身体，以加快这一进程，通过削减他们用针或手术刀（ 图4B，C）。吹打成的1.5 ml离心管中，避免将任何不溶解的组织取出鸡蛋。 注:使用为M任何微量离心管中根据需要来收集鸡蛋，并迅速工作，如卵的存活力将减少曝光的长时间来axenizing溶液。 </li><li>使用"高速设置"涡流管，持续15秒，以进一步除去附着在卵线虫组织。另外，如果一个涡流，则无法使用吸管上下溶液数次。 </li><li>沉淀蛋离心大约在18,000 g下2分钟。提高速度，如果鸡蛋没有足够的沉淀。除去axenizing溶液并用无菌蒸馏水和离心机填充离心管中再一次在900×g离心1分钟，冲洗两次。 </li><li>最后一次洗涤后，悬浮鸡蛋300-500微升的无菌蒸馏水，并将其放置在无菌3厘米培养皿或消毒手表玻璃。请记住，鸡蛋是现在表面消毒，所以用无菌吸管和/或pippetter提示和水，以保持蛋的清洁度。 </li><li>检查解剖显微镜（30-50X倍）下的蛋，以确认他们是否完好（ 图4D），并将它们转移到5cm的薄层板上，以肝肾阴虚型琼脂（参见2.1节） 注意:不要在板上增加超过400微升的卵悬浮液，因为它会使琼脂过度潮湿。 </li><li>标板的相关信息和在黑暗中直立存放在28℃。种蛋应该是很明显过夜。 注:上冷凝水盘的盖子，但术后1〜2天就会蒸发。在这个时候，将培养皿在塑料袋中，以保持在琼脂的水分并避免其干燥。 </li><li>定期观察菜肴和丢弃显示真菌或细菌污染的迹象，任何菜肴。一旦IJS被视为迁移了培养皿的壁，取下盖子，并用琼脂转让的底部培养皿，以修改后的白色陷阱12。转碟入modifie时，要考虑无菌条件d白平衡陷阱，以避免暴露介质的污染。 </li><li>继续监测白色陷阱和收获IJ需要。 注:Aposymbiotic线虫会继续入主陷阱的水中迁移了好几天，甚至几个星期。 </li></ol>

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没有利益冲突的声明。

使用合适的主机是成功的 EPN 体内饲养的关键因素。通常情况下，无论steinernematids和heterorhabditids可以复制，成功地完成其生命周期中的大蜡蛾， 大蜡螟 （鳞翅目:螟蛾科）幼虫。然而，其他的昆虫物种，从不同的家庭和/或单可以考虑。一些目前描述的线虫种类已知有特异性特定昆虫宿主。例如，S。 kushidai和S。 scarabaei都不是很致命的，以鳞翅目幼虫，需要鞘翅目...

昆虫病原线虫（EPN）， 斯氏和异小杆菌。 （斯氏，异小）及其共生细菌， 致病杆菌属和Photorhabdus菌（肠杆菌科）被认为是陆生动物，微生物的共生关系2-4,6,10,19。 嗜和Photorhabdus属一个新兴的模式。被包藏的共生体中的线虫的唯一自由生活阶段的肠，也被称为感染性（IJ）或第三阶段感染性8,10,13。细菌线虫对是致病性，适用范围广的昆虫，并已成功地在生物防治和综合虫害管理方案的全球6,8落实。 在这里我们将展示一系列的体内和被频繁运用的EPN的实验室条件下饲养体外技术， 我Ñ ​​体内方法考虑的昆虫宿主线虫的饲养。通常，各种昆虫的订单（ 即鳞翅目 ， 鞘翅目 ， 双翅目 ， 等 ）的未成熟阶段被认为是合适的宿主， 在体内方法通常被认为是对维护线虫培养物在实验室中。考虑到大规模生产的线虫时，这种方法可能不适合。可能需要用于此目的，要求更多的时间和相关的养虫额外成本大量昆虫宿主的。 昆虫致病性线虫，也可以在体外培养的几个媒体。根据不同的研究的目标; 体外方法可以或者考虑的共生细菌在媒体的掺入。在这份报告中，我们将介绍两种常用的方法EPN的传播。媒体的成份提供营养的共生杆菌的来源第二固醇源线虫。 体外方法提供了优势EPN的饲养没有昆虫宿主。 起初，许多体外媒体发展的被用于EPN的乘法时适合昆虫宿主不可用。然而，在过去几年中， 在体外饲养方法已被广泛应用在研究旨在了解EPN及其共生细菌17,19之间的互惠关系。 在此演示文稿详细的技术对应于那些由不同作者的库存实验室，亚利桑那州（亚利桑那州图森，美国）大学描述和细化。这些技术是从的技术，在大规模生产这些生物虫害管理的目的所使用的身体明显。

<table><tbody><tr><td colspan="4">For &lt;em&gt;In vivo&lt;/em&gt; Infections</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Filter paper, 9 cm grade 1 cellulose</td><td>Whatman/VWR</td><td>28450-081</td><td>Grade 1 filter paper recommended</td></tr><tr><td>Insect hosts</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>&lt;em&gt;Galleria mellonella&lt;/em&gt; are recommended</td></tr><tr><td colspan="4">For Liver-kidney Agar (for 500 ml)</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Beef liver, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Beef kidney, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Sodium chloride 2.5 g (0.5% final concentration)</td><td>Acros/VWR</td><td>200002-434</td><td>any research grade NaCl can be used</td></tr><tr><td>Agar, 7.5 g (1.5% agar, final concentration)</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>500 ml distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">For Lipid Agar (for 1 L)</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Nutrient broth, 8 g</td><td>BD/VWR</td><td>90002-660</td><td></td></tr><tr><td>Yeast extract, 5 g</td><td>EMD/VWR</td><td>EM1.03753.0500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate 10 ml (0.2 g/ml)</td><td>EMD/VWR</td><td>EM-MX0045-1</td><td></td></tr><tr><td>Corn oil, 4 ml</td><td>Any brand</td><td>Locally source; supermarket</td><td>Any brand of corn oil can be used</td></tr><tr><td>Corn syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H&lt;sub&gt;2&lt;/sub&gt;O and swirl for homogeneity</td><td>Karo</td><td>Locally sourced; supermarket</td><td></td></tr><tr><td>Agar, 15 g</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O, 890 ml</td><td></td><td></td><td></td></tr></tbody></table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

昆虫病原线虫（EPN）（ 斯氏和异小 ）与革兰氏阴性伽马变形菌的肠杆菌科细菌， 嗜菌与steinernematids线虫相关的家人互惠的伙伴关系，而Photorhabdus是heterorhabditids的共生体。一起线虫和细菌形成一个强有力的杀虫复杂，杀死了广泛的昆虫物种中的亲密和特殊的伙伴关系。在此，我们证明在体内和这些线虫在实验室条件下饲养常用的体外技术。此外，这些技术代表了成功建立EPN文化的关键步骤，并且还形成了基础，利用这些微生物为研究其它生物检测。的aposymbiotic（共生免费）线虫的生产往往是一个深入和多方面的方法来共生的研究是至关重要的。这协议不要求另外的抗生素，并且可以在时间与标准实验室设备很短的量来完成。以这种方式生产的线虫是比较稳健的，虽然根据所使用的种类及其在存储存活率可能会发生变化。在此演示文稿详细的技术对应于那些由不同作者的P.股票的实验室，亚利桑那大学（图森，亚利桑那州，美国）描述和细化。这些技术是从的技术，在大规模生产这些生物虫害管理的目的所使用的身体明显。

体内饲养方法使用活虫为线虫生长和繁殖的主机。感染室是用于曝光的昆虫IJS的有效方法。这是线虫的生命周期载体的细菌共生的一种昆虫主机之间的唯一舞台。 图1显示了设立感染室，以及建立该室所需要的材料， 在体外饲养方式让EPN成长没有昆虫宿主，但也实施了aposymbiotic（共生免费）线虫饲养。 图2显示了一个脂质琼脂平板上用不同的线虫阶段。脂质...

Watch this Scientific Journal Video about In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae at JoVE.com

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

本介绍的目的是在体内和体外技术用于昆虫致病性线虫的饲养证明， 在体内方法考虑这些线虫的饲养与昆虫宿主，而在体外方法利用丰富的琼脂培养基。

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

in-vivo-vitro-rearing-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Bioengineering

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

20.5K 次观看.  University of Arizona. 本介绍的目的是在体内和体外技术用于昆虫致病性线虫的饲养证明， 在体内方法考虑这些线虫的饲养与昆虫宿主，而在体外方法利用丰富的琼脂培养基。 

视频: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

科研

环境科学

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

遗传学

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

根结线虫的高和低通量筛选根结线虫。</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

免疫与感染

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

由Baermann Funnel快速分离野生线虫

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

生物学

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

培养和遗传操纵昆虫致病线虫

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

<meta charset="utf8"></head><body>优化松树线虫的感染

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are&#160;free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

<meta charset="utf8"></head><body>作者聚焦:用于研究宿主挥发性反应的松芽和松木线虫的体外共培养系统

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

在体内和昆虫病原线虫体外培育（斯氏和异小）

摘要

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此视频中的章节

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

根结线虫的高和低通量筛选根结线虫。

由Baermann Funnel快速分离野生线虫

使用荧光显微镜可视化中的细菌线虫

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

培养和遗传操纵昆虫致病线虫

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

松树线虫 Bursaphelenchus xylophilus 感染松树体内和体外及分离诱导挥发物

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

摘要

探索更多视频

此视频中的章节

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

根结线虫的高和低通量筛选根结线虫。

由Baermann Funnel快速分离野生线虫

使用荧光显微镜可视化中的细菌线虫

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

培养和遗传操纵昆虫致病线虫

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

松树线虫 Bursaphelenchus xylophilus 感染松树体内和体外及分离诱导挥发物

在体内和昆虫病原线虫体外培育（斯氏和异小）

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌